PROJECT SUMMARY Protein phosphorylation is an essential post-translational modification (PTM) that controls most biological processes. More than three-quarters of all proteins are phosphorylated at one or more sites in human cells. Systematic genome sequencing, gene expression and RNAi studies have implicated deregulation of kinase function in many human diseases, including cancer, diabetes, and neurodegeneration. However, such approaches do not reveal specific signaling pathways and molecular targets. Thus, there is an unmet need for the systematic interrogation of human kinase-substrate relationships. The long-term goal of our research is to decipher kinase signaling in basic biology and disease. To accomplish this, we have developed and applied quantitative phosphoproteomics strategies to connect specific kinases to their substrates, including for Polo- like kinase 1 (Plk1). Plk1 is the founding member of the Plk family and is conserved from yeast to humans. Plk1 is an essential regulator of recovery from DNA damage and mitotic entry, mitotic progression and cytokinesis, and is frequently overexpressed in cancer. While Plk1 is a bona fide oncogene, Plk2 and Plk3 act as tumor suppressors, protect cells against DNA damage, and are required for other G1 and S-phase processes, although the mechanisms that underlie these functions are largely unknown. Traditional strategies to selectively study kinase function such as gene deletion, depletion, or overexpression alter kinase abundance on a time scale of hours to days which often precludes assignment of direct kinase substrates. Elegant chemical genetics approaches that introduce mutations into the conserved catalytic kinase domain to render them ATP analog-sensitive have been implemented to overcome the general lack of selective inhibitors and the temporal control problem. However, these mutations often reduce kinase activity and stability, limiting the universal implementation of this approach. Thus, new strategies are needed for connecting kinases and their substrates. To address this gap in capability, we propose to establish a general quantitative chemical proteomics strategy to enable the identification of specific kinase substrates. Inducible protein degradation is an emerging technology for directly manipulating protein abundance. We hypothesize that the combination of inducible, rapid protein degradation (< 10 min half-life) and mass spectrometry based proteomics is a viable strategy for the identification of specific kinase substrates and elucidation of phosphorylation signaling networks of closely related enzymes. In this proposal, we provide a blueprint for comprehensive studies of kinase?substrate relationships on a kinome-wide level. This is pivotal for mapping cellular signaling pathways, identifying kinase pathway reprogramming upon disruption by mutations or drug treatment and resistance, and determining off-target effects of clinically relevant inhibitors. More than half of the human kinome is un- or under-characterized; experiments outlined here represent a roadmap for filling this gap in knowledge.